Pseudomonas aeruginosa ranks second among the most common human pathogens isolated from surgical sites, chronic and burn wounds. P. aeruginosa is also one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in biofilms makes it very difficult to eradicate the organisms by antimicrobial treatment. In order to eradicate P. aeruginosa biofilm infections, efforts should be focused on the developmental process leading to the formation of persistent and inherently resistant biofilms. Findings from our laboratory suggest that the two-component hybrid SagS is a key regulator of P. aeruginosa biofilm formation and biofilm cells transitioning to a highly antimicrobial resistant state. SagS was found to contribute to these two biofilm-specific regulatory circuits via two independent mechanisms. Biofilm formation required the hierarchical phosphotransfer-based signaling between SagS and the TCS BfiSR while biofilm tolerance was found to be dependent on BrlR, but independent of phosphotransfer, biomass accumulation, biofilm architecture, and the later stages of biofilm maturation, thus indicating SagS to be the regulator at which the two biofilm-specific regulatory circuits diverge. However, how SagS contributes to the activation of the two distinct developmental processes is not well understood. The goal of this project is to elucidate SagS signaling and regulatory events contributing to biofilm formation and biofilm cells transitioning to an antimicrobial tolerant state. The project i founded on the hypotheses that conserved amino acid (AA) residues present in the HmsP domain of SagS contribute to SagS promoting biofilm-specific regulatory circuits enabling biofilm development and/or biofilm tolerance and that interfering with or blocking the sensory function(s) of HmsP, via alanine substitution, will result in impaired biofilm development and/or biofilm tolerance. Experimentally, we will generate in Aim 1 SagS variants harboring alanine substitutions in conserved AA residues located in HmsP, the periplasmic sensory domain of SagS. sagS mutant biofilms expressing the resulting SagS-HmsPmutated constructs will be subsequently analyzed in Aim 2 by qRT-PCR for brlR expression. Mutant biofilms demonstrating altered or reduced brlR expression will then be analyzed for biofilm tolerance using antibiotic susceptibility and biofilm-MBC assays. Residues responsible for SagS contributing to biofilm development will be identified in Aim 3 by analyzing mutant cells for attachment, biofilm formation, and interactions of SagS- HmsPmutated with the TCS BfiSR. Findings from this detailed investigation will help to more completely define the mechanism by which SagS activates biofilm development and biofilm tolerance and how to manipulate SagS function(s).